Systemic mastocytosis (SM), a myeloproliferative disorder with variable clinical manifestations, is associated in most cases with the D816V mutation in KIT. The identification of the KIT D816V mutation in patients with systemic mastocytosis has gained a major prognostic significance in the last several years, largely because of the availability of tyrosine kinase receptor inhibitors such as imatinib. However, imatinib is ineffective in patients carrying KIT D816V mutation. In collaboration with Deciphera Pharmaceuticals, we thus evaluated inhibitors that target the switch pocket (SP) of KIT by altering the catalytic conformation of KIT, thus inhibiting mast cell proliferation and activation. Two SP inhibitors, DP-2976 and DP-4851, were examined for effects on neoplastic mast cell proliferation and mast cell activation. KIT D816V was blocked by these compounds in transfected 293T cells, HMC 1.1 and 1.2 human mast cell lines, and in CD34+-derived human mast cells activated by stem cell factor. Both inhibitors induced apoptosis in the neoplastic mast cell lines and reduced survival of primary bone marrow mast cells from patients with mastocytosis. Additionally, the SP inhibitors more selectively blocked SCF potentiation of FceRI-mediated mast cell activation. Overall, switch pocket inhibitors thus may provide therapeutic benefit. KIT has two major variants which differ by four amino acids (GNNK- or GNNK+) at the juxta-membrane region of the extracellular domain. We hypothesized that the expression pattern of these variants differ in systemic mastocytosis, and that transcripts containing the KIT D816V mutation segregate preferentially to one GNNK variant. A quantitative real-time PCR assay to assess GNNK- and GNNK+ transcripts from bone marrow mononuclear cells was developed. We found that the GNNK-/GNNK+ copy number ratio positively trended with % neoplastic mast cell involvement and KIT D816V containing transcripts displayed a significantly elevated GNNK-/GNNK+ copy number ratio. Relative expression of the GNNK- variant, but not the GNNK+, correlated with increasing percent of neoplastic mast cell involvement. A mast cell transfection system revealed that the GNNK- isoform of wild type KIT was associated with increased granule formation, histamine content and growth. These data suggest that neoplastic mast cells favor a GNNK- variant predominance, which in turn enhances the activating potential of the KIT D816V mutation and thus may influence therapeutic sensitivity in systemic mastocytosis.